Apparatus and method for the collection of interstitial fluids

ABSTRACT

The present invention involves apparatus and methods for use in collecting bodily fluids, such as interstitial fluids, from the epidermal layer of an animal. A preferred apparatus includes a pressure head and, optionally, a holder therefor for supplying a positive pressure to the head. The methods include the application of a positive pressure to the area surrounding an epidermal site from which stratum corneum has been breached, such as by laser ablation, to cause bodily fluids, such as interstitial fluids, to exude from the site and collecting the fluids exuding therefrom.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel apparatus and methods for the collectionof bodily fluids, such as interstitial fluids, from the body of ananimal, such as a mammal. The fluids so collected may then be analyzedfor biological or medical purposes, such as, for example, disease andhealth management activities. More particularly, this invention providesnovel apparatus and methods for the collection of large quantities ofinterstitial fluids from areas of the skin where the stratum corneum hasbeen breached.

2. Discussion of the Art

The stratum corneum is the outer horny layer of the skin comprising acomplex structure of compact keratinized cell remnants separated bylipid domains. In humans, the stratum corneum typically has a thicknessof about 10 μm to about 30 μm and overlays the epidermal layer, whichitself has a thickness of on the order of about 100 μm. The dermallayer, found below the epidermal layer, contains, among other things,capillary networks through which blood flows.

It has been proposed that interstitial fluids can be obtained from theepidermal layer in a minimally invasive procedure by stripping away thestratum corneum to expose the epidermal layer and thereafter collectinginterstitial fluids from the epidermis. Repeated application and removalof cellophane tape to the same location can be used to strip away thestratum corneum to expose the epidermal layer for the collection ofinterstitial fluids. Another technique available for the collection ofinterstitial fluids involves inserting a micro needle into the epidermallayer to allow fluids to be wicked up out of the body for deposit onto amembrane collection strip. This approach, however, requires preciseinsertion of the micro needle, oftentimes by trained medical personnel,and also results in biohazardous “sharps”.

Another series of techniques for collecting interstitial fluids aredescribed in PCT Patent Application, Serial No. PCT/US96113865,published on Mar. 6, 1997, International Publication No. WO97107734 andthe prior art cited therein (hereinafter referred to as the “PCTapplication”). The PCT application describes the use of energies atvarious wavelengths and frequencies to form micropores through thestratum corneum to a depth that exposes the epidermal layer. Methods toform such micropores include laser, sonic energy, and thermal energy,with or without the use of dyes or other energy absorbing materials toassist in the ablation and removal of the stratum corneum. In the PCTapplication, interstitial fluids are described as exuding from theepidermis after microporation of the stratum corneum. In addition, toinduce fluid flow, a vacuum (10 to 12 inches of Hg) can be applied tothe microporation sites (Examples 14 and 39 of the PCT applicationdescribed above). Example 14 describes the use of the recovered fluidsfor analysis of biological materials, such as glucose levels. In Example39, the use of a vacuum (i.e., a negative pressure) and ultrasound wassaid to produce an increase in the quantity of recovered interstitialfluid when compared with the use of vacuum alone.

In connection with the vacuum assist approach described in that PCTapplication, the volume collected is a function of the number ofmicropores, the level of vacuum, and the length of time the vacuum isapplied. However, the techniques disclosed in the PCT applicationreferred to above suffer from several disadvantages. First, even whenall variables are optimized, the quantity of interstitial fluidsobtained from the micropores in a short time period may not besufficient to utilize in various medically related testing procedures.Second, increasing the applied vacuum above about 13 inches Hg (about−6.5 psig) can result in visible hematomas of the skin and patientdiscomfort. Moreover, the use of vacuum assistance increases theevaporation of the fluids under extraction and requires a substantiallyair-tight seal around the microporation site, which can oftentimes bedifficult to achieve, even in a clinical setting. Finally, thistechnique also requires vacuum pumps and attendant fixtures, which canbe expensive to acquire and maintain.

These and other disadvantages of the prior art are overcome by theapparatus and method of the present invention. In particular, thepresent invention provides apparatus and methods that allow thecollection of large quantities of bodily fluids, such as interstitialfluids, from the epidermal layer over short periods of time, whencompared with the amounts collectable through prior art techniques,without the need for vacuum assist devices. The apparatus and methodsare inexpensive to fabricate, easy to use, and present minimaldiscomfort to the patient.

SUMMARY OF THE INVENTION

In the present invention, it has been discovered that increased amountsof interstitial fluids can be collected from micropores formed throughthe stratum corneum and extending into the epidermal layer by using anovel cup-shaped pressure head applied to the area of the skinsurrounding the micropores. The pressure head is applied under apositive pressure, the force of which may fall within the broad range ofabout 1 to about 11 pounds, preferably from about 3 to about 11 pounds,with about 4 to about 9 pounds being preferred. The pressure headincludes an aperture of diameter sufficient to surround the micropores,together with a reservoir volume in which the fluids may be collectedand maintained and from which the fluids may be sampled or removed. Thepositive pressure may also be conveniently applied using the pressurehead, with collection of fluids being carried out with separateapparatus, such as a capillary tube, an absorbent material, or othersuitable device. The head may be housed in a holder having an air ram orother mechanism to provide variable pressure to the head when the headis placed on a patient's skin. The method of the present inventionincludes forming a breach through the stratum corneum and into theepidermal layer, followed by the application of a positive pressure tothe area surrounding the microporation site to cause interstitial fluidsto exude therefrom. The interstitial fluids are then collected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a typical microporation site having six micropores;

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G are cross-sectional views ofpressure heads of the present invention;

FIG. 3 is a graph of the data derived from Example 2;

FIG. 4 is a graph of the data derived from Example 2;

FIG. 5 is a graph of the data derived from Example 2;

FIG. 6 depicts the holder and pressure head arrangement of the presentinvention;

FIGS. 7A through 7J are graphs of the data derived from Example 3;

FIGS. 8A through 8C are graphs of the data derived from Example 4;

FIGS. 9A through 9H are graphs of the data derived from Example 5; and

FIGS. 10A and 10B are schematic views of an apparatus that can be usedto apply a force to the skin to aid in the collection of interstitialfluids therefrom.

FIGS. 11A and 11B are schematic views of an apparatus that can be usedto apply a force to the skin to aid in the collection of interstitialfluids therefrom.

DETAILED DESCRIPTION

The present invention makes use of a pressure head that is positioned onthe skin of an animal, such as a mammal, in a manner to encompass a sitethat has first been treated to breach the stratum corneum.Advantageously, the pressure head can be used in instances in which thestratum corneum has been removed by microporation techniques to exposethe epidermal layer. Such microporation techniques are described indetail in the PCT application referred to above, which is incorporatedherein by reference.

For example, the microporation technique may involve the use of focusedlaser energy of a power and pulse width sufficient to ablate the stratumcorneum to expose the epidermal layer without substantial exposure ofthe dermal layer. This technique may be used with dyes or other energyabsorbing materials to assist in the transfer of energy to the stratumcorneum, and hence ablation of the stratum corneum, or may be usedwithout such absorbing materials and may be applied to form one or moremicropores, either sequentially or concurrently. Such micropores may beof circular, elliptical, or other shape. As used herein, the term“micropore” means a small breach or pore formed in the stratum corneumin a selected area of the skin to lessen the barrier properties of thestratum corneum such that fluids, for example interstitial fluids, canexude from the epidermal layer. Such micropores include those describedin the PCT application referred to above and also include openings orbreaches through the stratum corneum having diameters of on the order ofup to 500 μm, with about 100 μm being preferred. For example, FIG. 1shows a typical microporation site that includes six micropores 12, eachhaving an elliptical shape of about 80 μm by 100 μm in size. The overallsize of the microporation site is about 1.5 mm when measured from theouter edges of the micropores 12. The centers of micropores 12 of FIG. 1lie on a circle having a diameter of about 1 mm, with the centers ofadjacent micropores being about 450 μm apart.

Prior art techniques for collecting interstitial fluids from amicroporation site, such as site 10 of FIG. 1, involve either collectingthe fluids as they naturally exude from the site or by providing avacuum (i.e., negative pressure) to the site to cause more fluids toexude from the micropores 12. While these techniques permit thecollection of some quantities of interstitial fluids, it has beendiscovered that significantly larger quantities of fluids can becollected in a shorter amount of time using the apparatus and methods ofthe present invention.

In particular, it has been discovered that the topical application of apositive pressure to the area surrounding the microporation site 10permits recovery of interstitial fluids in an amount that is from aboutthree (3) to about thirty (30) times or more than the amounts collectedusing the vacuum assist technique described above and in the PCTapplication referred to herein.

It has also been discovered that the positive pressure canadvantageously be applied by using a generally cup-shaped pressure headthat may be included in a holder that permits the application ofvariable amounts of positive pressure to the microporation site.

Thus, the present invention described herein can be utilized for thecollection of interstitial fluids from a microporation site,irrespective of the techniques used to form the breach. Although theexamples which follow below describe the use of the present apparatusand methods to collect fluids from micropores formed via laser energy,the invention is not so limited.

Referring now to FIGS. 2A through 2G, wherein like reference numeralsrefer to like components, various pressure heads of the presentinvention are generally depicted. The heads may be made from anysuitable polymeric material, such as, for example acrylic,polypropylene, polyethylene, and others, including copolymeric andterpolymeric materials, as well as suitable metallic materials such asstainless steel, or such other materials suitable for formation of thehead and application of pressure to the skin.

FIG. 2D depicts a pressure head 14 having at one end thereof a threadedend 16; preferably the threads are on the exterior wall 18 of the head14, although the threads may also be along the interior wall 20. Theinterior of the head 14 forms a reservoir 22. The head 14, at the endopposite threaded end 16, includes a bottom portion 24 which may becircular, elliptical, square, rectangular or other shape. An aperture 26is formed through the portion 24 to form a communication channel to thereservoir 22. In the head 14 of FIG. 2D, which is referred to in theexamples that follow as “Head D”, the radius of curvature 28 of theexterior wall 18 near the bottom portion 24 is 0.45 inches (11.43 mm),the bottom portion 24 being circular and having a diameter of 0.25inches (6.35 mm), and the aperture being circular and having a diameterof 0.10 inches (2.54 mm). As explained hereinafter, it has beendiscovered that the radius of curvature of the exterior wall 18 near thebottom portion 24 has an effect on the quantities of interstitial fluidsthat can be collected from a microporation site.

FIG. 2B depicts a pressure head 14, referred to below as “Head B”, whichis similar to that shown in FIG. 2D; however, the bottom portion 30 ofthe pressure head of FIG. 2B is concave and has a radius of curvature 32of 0.50 inches (12.7 mm), with the concave portion having a diameter of0.311 inches (7.9 mm).

FIG. 2C depicts a pressure head 14, referred to below as “Head C”. Theexterior wall 18 of Head C has a radius of curvature 34 of 0.75 inches(19.05 mm) and the interior wall 30 of Head C has a corresponding radiusof curvature 36 of 0.650 inches (16.51 mm). The bottom portion 24 of thehead 14 of FIG. 2C is circular and has a diameter of 0.25 inches (6.35mm).

FIG. 2A depicts pressure head 14, referred to below as “Head A”. Theexterior wall 18 of Head A has a radius of curvature 38 of 0.45 inches(11.43 mm) and the interior wall 20 of Head A has a radius of curvature40 of 0.361 inches (9.17 mm). The bottom portion 24 of the head 14 ofFIG. 2A has a diameter of 0.377 inches (9.58 mm).

FIG. 2E depicts pressure head 14, referred to below as “Head E”. Thebottom portion 24 of the head 14 of FIG. 2A is circular and has adiameter of 9.5 mm.

FIG. 2F depicts pressure head 14, referred to below as “Head F”. Thebottom portion 24 of the head 14 of FIG. 2F has a diameter of 9.5 mm.

FIG. 2G depicts pressure head 14, referred to below as “Head G”. Thebottom portion 24 of the head 14 of FIG. 2G has a diameter of 5.7 mm.

In the most general sense, the method of the present invention includesthe steps of forming a breach through the stratum corneum and into theepidermal layer, followed by the application of a positive pressure tothe area surrounding the microporation site to cause interstitial fluidsto exude therefrom. The interstitial fluids are then collected. Thefluids can then be analyzed to determine the concentration of ananalyte, such as glucose. In operation, one specific method for thecollection of interstitial fluids from the body of an animal comprisesthe steps of:

(a) forming a breach through the stratum corneum of the animal, suchthat the breach extends at least into the epidermal layer of the skin ofthe animal;

(b) placing a pressure head adjacent to the breach;

(c) exerting a positive pressure on the pressure head in a directiongenerally toward the skin of the animal; and

(d) collecting fluids from the breach. In this specific method, it ispreferred that the pressure head be positioned such that the fluids flowthrough the aperture in the pressure head and into the reservoir.

Alternatively, another specific method for the collection ofinterstitial fluids from the body of an animal comprises the steps of:

(a) placing a pressure head against the skin of the animal;

(b) forming a breach through the stratum corneum of the animal such thatthe breach extends at least into the epidermal layer of the skin of theanimal, the breach being adjacent to said pressure head;

(c) exerting a positive pressure on the pressure head in a directiongenerally toward the skin of the animal; and

(d) collecting fluids from the breach.

A positive pressure can be exerted on the pressure head prior to formingthe breach in the stratum corneum. In this specific method, it ispreferred that the breach be formed so that it is in register with theaperture in the pressure head so that the fluids flow through theaperture in the pressure head and into the reservoir. Variations ofthese specific procedures can also be used.

In order to examine the efficiency of interstitial fluids collectionusing the pressure heads and methods of the present invention, a seriesof tests were performed, as described below.

EXAMPLE 1

In this example, nine (9) human volunteers were used. The interiorforearms (between the elbow and the wrist) of each volunteer weresubject to laser microporation (wavelength of 810 nm, 20 millisecondpulse width, approximately 250 milliwatts, 20 to 30 pulses applied,black tape applied to the skin to act as an energy absorber) to form amicroporation site similar to microporation site 10 shown in FIG. 1. Twosuch sites were made on each arm of each subject and were hydrated witha water droplet placed on the microporation site for 10 to 15 seconds,followed by drying (using gentle blotting) prior to fluid extraction.Thus, a total of four microporation sites were made on each subject.

One site on the right arm and the corresponding site on the left arm ofeach subject were treated in the following manner. Head D, FIG. 2D, wasmanually placed over the microporation site so that the aperture 26encompassed the site. Manual pressure was exerted on Head D in adirection toward the microporation site for sixty (60) seconds.Interstitial fluids flowed into Head D and were collected by means of 1μl capillary tubes, with the collected volume recorded. By means of thistechnique, the volume of interstitial fluids recovered ranged from 0.34to 1 μl.

The second site on the right arm and the corresponding second site onthe left arm of each subject were treated as follows. A vacuum system,−7.5 psig (15 inches of Hg), was applied to each microporation site for60 seconds. Interstitial fluids were observed to flow from themicroporation site and were collected by means of 1 μl capillary tubes,with the collected volume recorded.

The interstitial fluids (hereinafter “ISF”) collected by the applicationof positive pressure were thereafter analyzed for glucose levels. It isto be noted that for each use of the vacuum assist technique, the volumeof interstitial fluids recovered was in the range of 0.1 to 0.2 μl andno glucose determination was made. As a control, a finger stick was alsoperformed on each volunteer and approximately 50 μl of blood waswithdrawn. The blood samples were centrifuged and the plasma analyzedfor glucose values. Table 1 below presents the results of this example.

TABLE 1 Volunteer ISF Glucose ID * collected (μl) in sample (mg/dl) JK-10.73 153.29 JK-2 0.74 135.27 JK-3 — 124.4 NL-1 0.77 147.01 NL-2 1.36150.00 NL-3 — 129.6 SW-1 0.36 260.56 SW-2 0.34 275.88 SW-3 — 270 DS-10.34 126.76 DS-2 0.50 136.60 DS-3 — 115.9 TM-1 0.63 124.60 TM-2 0.87124.94 TM-3 — 126.1 ML-1 1.00 96.00 ML-2 0.45 111.33 ML-3 — 103.5 JG-10.89 126.97 JG-2 1.35 111.04 JG-3 — 104.1 GH-1 0.92 90.43 GH-2 0.5393.21 GH-3 — 91.1 KN-1 0.69 103.91 KN-2 0.68 113.24 KN-3 — 94.8 *Thenumber following each volunteer ID represents the following: 1 = leftarm, pressure applied for 60 seconds; 2 = right arm, pressure appliedfor 60 seconds; and 3 = finger stick sample for comparison.

As can be seen from the foregoing table, the glucose values measuredfrom the collected ISF are reasonably correlated to the glucose valuesobtained from the blood plasma. This example also demonstrates that thevolume of ISF that can be obtained by the positive pressure techniquedisclosed herein is significantly greater than that obtained when usingthe vacuum method.

EXAMPLE 2

In this example, the sequential application of pressure followed byvacuum and vacuum followed by pressure was investigated. Twomicroporation sites, similar to FIG. 1, were made on each arm (left andright) of seven (7) human volunteers by means of the technique ofExample 1. The microporation sites were hydrated as in Example 1 andwere treated as follows. To one site on one arm (e.g., the right arm),Head D was applied, under manual pressure, for 60 seconds, ISF wascollected, and the volume recorded. Thereafter, within two to fiveminutes, a vacuum system was used to apply a vacuum (13 inches Hg) tothe same site for 60 seconds, ISF was collected, and the volumerecorded. This pressure/vacuum technique was then applied to thecorresponding site on the volunteer's left arm. To the second site onthe first arm (e.g., the right arm) the vacuum system was first applied(13 inches Hg) for 60 seconds, ISF was collected, and the volumerecorded. Thereafter, within two to five minutes, Head D was applied,under manual pressure, for 60 seconds to the same site, ISF wascollected, and the volume recorded. This vacuum/pressure technique wasthen applied to the corresponding site on the volunteer's left arm inthe same manner. Table 2 below presents the results of this example.

TABLE 2 Volume ISF Volume ISF (μl) collected (μl) collected Volunteer IDArm Condition* under pressure under vacuum MP Right P/V 0.79 0.18 LeftP/V 1.25 0.11 Right V/P 0.61 0.03 Left V/P 1.00 0.08 JG Right P/V 1.090.21 Left P/V 1.12 0.19 Right V/P 1.26 0.07 Left V/P 1.52 0.05 KN RightP/V 0.45 0.34 Left P/V 0.35 0.34 Right V/P 0.90 0.10 Left V/P 0.60 0.19PB Right P/V 0.53 0.30 Left P/V 0.35 0.27 Right V/P 0.44 0.06 Left V/P0.61 0.02 NL Right P/V 0.75 0.30 Left P/V 1.15 0.36 Right V/P 0.91 0.12Left V/P 1.18 0.26 DS Right P/V 0.48 0.25 Left P/V 0.27 0.33 Right V/P0.38 0.10 Left V/P 0.63 0.07 JB Right P/V 0.71 0.38 Left P/V 0.48 0.48Right V/P 0.85 0.15 Left V/P 1.35 0.12 P/V = Pressure followed byvacuum; V/P = Vacuum followed by pressure

As noted in Table 2, except for one instance involving volunteer DS, theapplication of pressure gave a greater volume of ISF than did vacuum,irrespective of whether the pressure was applied before or after thevacuum. These data are presented graphically in FIG. 3.

FIG. 4 presents these data in a slightly different form. As there shown,for example for volunteer MP, the average volume of ISF recovered fromthe right arm through the application of pressure is shown in the leftmost bar as 0.70 μl. This value is obtained from the foregoing table,where the ISF collected by the pressure technique is 0.79 μl and 0.61 μlfrom the right arm; the average is 0.70 μl. The remaining data found inFIG. 4 is determined in the same manner. FIG. 4 thus highlights that theuse of positive pressure to obtain ISF is superior to vacuum techniques.

FIG. 5 is a further depiction of the data of Table 2 and again shows thedistinct advantages of using positive pressure to obtain ISF. As thereshown, the data are grouped by volunteer, according to the method firstapplied to collect ISF. For example, the left most bar in each data setrepresents the average volume of ISF collected from both arms of eachvolunteer during the 60 seconds of pressure application when pressure isapplied first. For volunteer MP, this average value of 1.02 μl isobtained from the Table 2 data for the right and left arms (i.e., 0.79μl and 1.25 μl respectively). The second bar represents the averagevalue of ISF collected from both arms of volunteer MP during the 60seconds of vacuum application when pressure is applied first. The thirdbar represents the average value of ISF collected from both arms ofvolunteer MP during the 60 seconds of vacuum application when vacuum isapplied first, followed by pressure. Finally, the fourth bar representsthe average value of ISF collected from both arms of volunteer MP duringthe 60 seconds of pressure application when vacuum is applied first. Thedata depicted in FIG. 5 for the remaining volunteers is obtained in asimilar manner.

The means of the data set forth above in Table 2 also show that theapplication of positive pressure provides significant advantages to thecollection of ISF over the vacuum technique. The following Table 3 setsforth the means of this data.

TABLE 3 Mean ISF volume collected Mean ISF volume collected from botharms during 60 from both arms during 60 seconds of pressure seconds ofvacuum Volunteer ID application (μl) application (μl) MP 0.91 0.10 JG1.25 0.13 KN 0.57 0.24 PB 0.48 0.16 NL 1.00 0.26 DS 0.44 0.19 JB 0.850.28

Thus, for each volunteer, the use of positive pressure providedsignificantly higher ISF collection volumes than could be obtained fromthe use of vacuum. Indeed, across all volunteers, the mean collectionvolume of ISF was 0.78 μl by pressure but only 0.19 μl by vacuum, adifference of over 300%.

EXAMPLE 3

As a follow-up to Examples 1 and 2, a further set of studies wasperformed on five (5) human volunteers. In these studies, microporationsites, similar to FIG. 1, were formed on the interior forearm of thevolunteer by means of the technique of Example 1; the sites werehydrated as in Example 1. Head D (see FIG. 2D) was used as the pressurehead and was attached to the holder 42 shown in FIG. 6. That holderincludes a base plate 44 having a threaded opening 46 for engagementwith the threaded end 16 of head 14 (shown in dotted lines in FIG. 6).The holder 42 also includes a movable vertical plate 48 attached to thebase plate 44. The movable plate 48 is connected to a ram 50. The ram50, which may be an air driven or hydraulic ram or a biased spring ram,operates to exert a force on the base plate 44, and hence to thethreaded end 16 of the head 14. The ram 50, as depicted in FIG. 6, iscoupled to a top plate 52, which in turn is coupled to a stand 54. Ofcourse, other ways of connecting the ram 50 to the movable plate 48 canbe used. The stand 54 may also be provided with a tongue (or groove) orother suitable mechanism for engagement with a groove (or tongue) orother suitable mechanism on the movable plate 48, as generally depictedby dotted lines 56 in FIG. 6. Such arrangement permits the movable plate48 to travel in a repeatable manner when the holder 42 is used. The rammay exert a known force to the head 14, which force may be varied fromone use of the holder to another or during any single use thereof. Inthis Example 3, the holder was operated such that a force of 4 through11 pounds could be applied to the head 14 at the threaded end 16thereof.

During the course of Example 3, the force applied to the threaded end 16of Head D was maintained constant during any single run, but was variedfrom one run to the next. Thus, the following description of the testsperformed on Subject 1 applies to the remaining subjects, unlessotherwise noted.

After formation of the microporation site and hydration as in Example 1,Head D, having a circular bottom portion 24 with a diameter of 2.5 mm,was applied to the microporation site using a force of 5 pounds on thethreaded end 16. The ISF flux (in μl/minute) was then measured in 30second increments over an elapsed time of 6 minutes. Thereafter, Head Dwas removed from the microporation site. A new microporation site wasformed and hydrated as in Example 1 and Head D was applied to this newsite using a force of 6 pounds on the threaded end 16. The ISF flux (inμl/minute) was measured as described, after which Head D was againremoved. Another microporation site was formed and the above procedurewas repeated using a force of 7 pounds applied to the threaded end ofHead D. The procedures were again repeated, as described, with theapplication of 8 and 9 pounds of pressure to the threaded end of Head D.Subjects 2 and 3 were treated as described above. Subject 4 was treatedin the same manner, except that a force of 11 pounds on the threaded end16 of Head D was also studied. Subject 5 was also treated in the samemanner, except that the forces applied to the threaded end 16 of Head Dwere 4, 5, 6, 7 and 8 pounds.

This example also investigated the effect on ISF recovery caused byincreasing the diameter of the bottom portion 24 of Head D. Thus, theprocedures described above were used in conjunction with the Head D ofFIG. 2D in which the diameter of the bottom portion 24 was 3.0 mm.

FIGS. 7A through 7J depict the results of this example, in which theflux rate of ISF is plotted against time (in minutes) for the appliedforces and where the figures represent the diameters of the bottomportion 24 of Head D as described in the following Table 4.

TABLE 4 Subject 2.5 mm diameter 3.0 mm diameter 1 FIG. 7A FIG. 7B 2 FIG.7C FIG. 7D 3 FIG. 7E FIG. 7F 4 FIG. 7G  FIG. 7H* 5 FIG. 7I FIG. 7J*Note: 11 pounds pressure not studied

Referring to FIGS. 7A, 7C, 7E, 7G, and 7I, it will be noted that, inmost instances for each force applied, the rate of ISF flow increasesfor the first 60 seconds that the force is applied and then tends todecrease thereafter. However, there are some variations from subject tosubject and, to a more limited extent, within the subjects themselves.The same general observations can be made from FIGS. 7B, 7D, 7F, 7H, and7J.

Comparing the results obtained from using the 2.5 mm diameter Head D tothose from using the 3.0 mm diameter Head D, it can be seen that for allsubjects, except Subject 4, the initial rate of ISF flow was greater forthe 2.5 mm diameter Head D.

EXAMPLE 4

In this example, Subjects 1, 3, and 6 of Example 3 were used to test therecovery rate of the ISF using a vacuum followed by the application ofpositive pressure. A microporation site was prepared and hydrated bymeans of the technique of Example 1 and the volume of recovered ISF wasmeasured. For these subjects, ISF was collected for 120 seconds usingvacuum (−12.73 psig), immediately followed by vacuum removal for 60seconds for site recovery. After recovery, vacuum (−12.73 psig) wasagain applied for 120 seconds, followed by 60 seconds of site recovery(vacuum removed). This procedure was repeated five times using vacuumassistance. Each subject was then allowed a five minute recovery period,following which ISF was collected for 120 seconds using Head C with aforce of 7 pounds applied to the threaded end 16 of Head C. Thereafter,Head C was removed from the microporation site for 60 seconds to allowfor recovery. At the end of the collection period, ISF collection wasperformed for 120 seconds using Head C with a force of 7 pounds appliedto the threaded end 16 of Head D. After this collection, Head C wasremoved for another recovery period of 60 seconds. Collection in thismanner using positive pressure was carried out five times over theperiod.

The results of this example are shown in FIGS. 8A through 8C. From FIGS.8A through 8C, it is noted that the volume of ISF collected from eachsubject using the vacuum approach remained substantially constant overthe test period, although the volume collected from Subject 3 decreasedat 8 minutes and 10 minutes.

On the other hand, the volume collected from each subject upon theapplication of positive pressure generally decreased over the entiretest period of 10 minutes. It is thus theorized that the ISF in theepidermis exists in equilibrium with fluids in the underlying dermallayer and the surrounding tissues. Removing large quantities of ISF fromthe epidermis and dermis over a relatively short period of time, withoutproviding a sufficient recovery period, upsets this equilibrium anddepletes the ISF residing in the epidermis and dermis of the treatedarea. Indeed, in connection with the present invention, it has beenobserved that when a recovery period of on the order of 3 to 8 minutesis used, the next removal of ISF by application of positive pressurewill be of a high volume. For example, with reference to FIG. 8A, hadthe recovery time between the 6 minute and 8 minute positive pressuredata been longer than the 60 seconds used, the volume of ISF recoveredat 8 minutes would have been of the magnitude shown for the 2 minutepressure data. This result has led to the theory that there twomechanisms affecting the quantity of ISF in the epidermis. First, ISFnaturally resides in the epidermal and dermal layers and is available tobe removed upon the application of pressure. Second, there is a steadyinflux of ISF consisting of blood plasma filtrate from the capillaries,through the dermal layer, and into the epidermis. Although this influxoccurs at a finite rate, this observation establishes the ability tocontinuously monitor ISF for fluid analysis and other purposes.

EXAMPLE 5

In this example, Heads A through D (see FIGS. 2A-2D), each having anaperture diameter of 2.5 mm, were used to collect ISF from Subject 1 ofExample 3. A microporation site was prepared by means of the techniqueof Example 1 and ISF was collected by means of the technique describedin Example 3. The rate of ISF removal and the volume of ISF removed wasmeasured. FIGS. 9A through 9H set forth the flux rate and cumulativerecovered volume data obtained in this example. As seen from acomparison of FIGS. 9A, 9C, 9E, and 9G, the Head C generally providesthe greatest initial flow (i.e., slope) of ISF over the first 60 secondsas compared to Heads A, B and D. As observed from FIGS. 9B, 9D, 9F, and9H, the Head C also provides the greatest volume of ISF collected overall applied pressures as compared to the other heads.

These data thus indicate that the head shape, particularly the radius ofcurvature, has an effect on the flow rate and volume of ISF recovered.In particular, as the shape of the exterior wall 18 of the pressure head14 approaches that of a cylinder (i.e., no curvature along thelongitudinal axis of the head when viewed from the threaded end 16 tothe bottom portion 24), the rate of ISF flow and the volume of ISFrecovered increases.

EXAMPLE 6

In this example, six different head configurations were used to extractISF. Twelve microporation sites, similar to those of FIG. 1, were madeon the interior forearms of five human volunteers by means of thetechnique of Example 1. Heads were attached to the holder 42 shown inFIG. 6. Head A was attached to the holder and was used to apply four (4)pounds force to the microporation site in the same manner as in Example3. Another microporation site was then formed and head A was used toapply six (6) pounds of force to the microporation site. This processwas repeated in this fashion until all heads, A B, C, D, E, and F wereused on each subject. In each case, the ram fixture was used as inExample 3. ISF flux (in μl/minute) was then measured in 30-secondincrements over a five-minute period.

Table 5 shows the results of this experiment, including the averageamount of ISF collected for all five subjects, for the times of 30seconds and 60 seconds. Table 5 also shows the percentage of the totalamount of ISF collected in 60 seconds that was collected in the first 30seconds. This percentage indicates how quickly the rate of collectionincreases to its maximum and is pertinent because it is desirable forthe instrument to collect the fluid in a short amount of time.

TABLE 5 Percent of Average Median fluid volume volume collected Percentof Percent of Force collected Std. Dev. RSD* collected in first 30collections collections Head (pounds) (μl) (μl) (%) (μl) seconds >1μl >0.5 μl A 4 0.23 0.22  96 0.16 12  0 20 A 6 0.41 0.37  89 0.45 31  040 B 4 0.15 0.18 115 0.08 29  0  0 B 6 0.11 0.13 112 0.08  6  0  0 C 40.52 0.40  77 0.35 35 20 40 C 6 0.93 0.29  32 1.10 39 60 80 D 4 0.380.40 103 0.23 28 20 20 D 6 0.48 0.25  51 0.44 34  0 40 E 4 1.03 0.24  231.02 44 60 100  E 6 1.44 0.48  34 1.42 44 80 100  F 4 1.12 0.43  38 0.9843 40 100  F 6 1.92 0.43  23 2.07 43 100  100  *RSD means standarddeviation (Std. Dev.) divided by average volume collected times 100%.Total amount of fluid collected (μl): Subject 1: 10.72 Subject 2:  9.39Subject 3:  8.45 Subject 4:  4.49 Subject 11: 10.66

EXAMPLE 7

In this example, 18 microporation sites, as in Example 1, were made onthe interior forearms of six human volunteers. In this example, themicropores were arranged (a) singly, (b) in a straight line separated by1 mm, or (c) in a triangle with each micropore forming a vertex of anequilateral triangle 1 mm on each side. Heads C, E, and G were used. Theforce was either four (4) pounds or seven (7) pounds for eachcombination of pore number and head. A fixture similar to the ramfixture was used, but instead of compressed air, this fixture utilized asystem of weights applied to the top of the ram to deliver the force.This change was made to increase the accuracy of the force delivery andto reduce friction in the force delivery device. Fluid was collected forone minute at intervals of 30 second (if possible) and the volumecollected was calculated.

Table 6 below shows the results of this example, including the averageamount of ISF collected for all five subjects, for the times of 30seconds and 60 seconds. Table 6 also shows the percentage of the totalamount of ISF collected in 60 seconds that was collected in the first 30seconds. This percentage indicates how quickly the rate of collectionincreases to its maximum and is pertinent because it is desirable forthe instrument to collect the fluid in a short amount of time. The finalcolumn shows the increase in volume of fluid collected in one minute inthe presence of additional micropores relative to volume of fluidcollected in the presence of a single micropore. In general terms, thepercentage increase in going from one micropore to two micropores wasgreater than the percentage increase in going from two micropores tothree micropores. This was especially true of the more aggressive heads(head E and head G).

TABLE 6 Ratio of amount Percent collected of fluid compared Averagecollected to amount volume in first Percent of Percent of collectedForce Number collected Std. RSD* 30 collections collections with 1 Head(pounds) of pores (μl) Dev. (μl) (%) seconds >1 μl >0.5 μl pore C 4 10.47 0.24 51 45  0 50 1.00 C 4 2 0.80 0.31 39 38 20 80 1.70 C 4 3 1.000.43 44 42 60 80 2.10 C 7 1 0.88 0.42 48 47 20 80 1.00 C 7 2 1.06 0.2321 47 50 100  1.20 C 7 3 1.57 0.47 30 52 80 100  1.80 E 4 1 0.79 0.22 2844 10 90 1.00 E 4 2 1.27 0.47 37 40 60 100  1.60 E 4 3 1.86 0.58 31 43100  100  2.30 E 7 1 1.22 0.22 18 53 90 100  1.00 E 7 2 2.29 0.51 22 54100  100  1.90 E 7 3 2.99 0.57 19 48 100  100  2.50 G 4 1 1.75 0.41 23100  100  1.00 G 4 2 2.50 0.38 15 100  100  1.40 G 4 3 2.58 0.47 18 100 100  1.50 G 7 1 2.59 0.34 13 100  100  1.00 G 7 2 3.04 0.59 19 100  100 1.20 G 7 3 3.51 0.62 18 100  100  1.40 *RSD means standard deviation(Std. Dev.) divided by average volume collected times 100%. Total amountof fluid collected (μl): Subject 1: 40 Subject 2: 55 Subject 4: 46Subject 8: 44 Subject 11: 45

EXAMPLE 8

This example shows the effect of aperture diameter on the amount offluid collected and the rate at which [the greatest percentage of fluid]is recovered. In this example, 11 head configurations were tested on theinterior forearm of five subjects. The configurations were as follows:

Diameter of aperture Head (mm) C 1.5 C 2.5 C 3.0 C 4.0 E 1.5 E 2.5 E 3.0E 4.0 G 1.5 G 2.5 G 3.0

Three micropores were arranged in a triangle, with each microporeforming a vertex of an equilateral triangle 1 mm on each side. Fourpounds of force was used for each extraction. Each extraction had aduration of 60 seconds, with samples being collected at 30 and 60seconds.

Table 7 shows the results of this experiment, including the averageamount of ISF collected for all five subjects, for the times of 30seconds and 60 seconds. Table 7 also shows the percentage of the totalamount of ISF collected in 60 seconds that was collected in the first 30seconds. This percentage indicates how quickly the rate of collectionincreases to its maximum and is pertinent because it is desirable forthe instrument to collect the fluid in a short amount of time. Thisexample shows that varying the diameter of the aperture at the center ofthe head can result in significant changes in the volume of fluidcollected and flux rates. The smaller the aperture, the faster the ISFis collected, but a lower total volume is collected. At the largestaperture tested (4 mm), the fluid flux rate had significantly decreased,and total volume of ISF collected differed significantly from that whenoptimum size was used. The optimum size was found to be 2.5 mm to 3.0 mmwith these head configurations.

The conditions for forming the micropores in the skin and applying theforce to the skin were as follows:

1. Dye #5, ICI 2 mil w/carbon, removed after poration

2. Umbilical porator, 30 pulses, 250 mw

3. 30 ms pulse, 60 ms delay

4. “direct” RAM with 4 lbs. of weight

TABLE 7 Average Average Percent of Inside volume volume fluid diametercollected, collected, collected Percent of Percent of of 0-30 sec RSD*0-60 sec RSD* in first 30 collections collections Head aperture (μl) (%)(μl) (%) seconds >1 μl >0.5 μl C 1.5 0.1033 74 0.2809 49 37  0  0 C 2.50.1828 70 0.5363 62 33 10 30 C 3.0 0.1606 70 0.5106 48 30 10 40 C 4.00.1225 50 0.4416 32 27  0 30 E 1.5 0.2729 54 0.6906 37 37 10 80 E 2.50.4131 54 1.0747 43 35 70 80 E 3.0 0.4369 41 1.2369 33 34 70 100  E 4.00.3803 47 1.2038 31 31 70 100  G 1.5 1.2344 30 1.9556 18 63 100  100  G2.5 1.6919 14 2.5638 11 66 100  100  G 3.0 1.7703 20 2.6272 17 67 100 100  *RSD means standard deviation (Std. Dev.) divided by average volumecollected times 100%. Total amount of fluid collected (μl): Subject 1:21 Subject 4: 25 Subject 6: 26 Subject 8: 28 Subject 11: 31

Pressure can be applied to the skin by means of apparatus other than thepressure head and ram previously described. FIGS. 10A and 10B show anapparatus that employs a vacuum to cause atmospheric pressure to actupon a piston in a cylinder and cause it to apply a force to the skin.The apparatus allows the user, i.e., the patient, to apply force to abody part, such as a forearm, without the need for providing an opposingforce to inhibit motion.

Typically, when pressure is employed to force interstitial fluids toexude from the skin, a stopping mechanism is required to oppose theapplied force and keep the body part stationary. An apparatus thatexerts a force on the skin of the forearm normally requires a means forsupporting the backside of the arm, typically through the use of amechanical clamp or an immovable object, such as a table. These meansare large and uncomfortable for the user, or they require propertechnique to provide consistent results. The apparatus shown in FIGS.10A and 10B can be made in small sizes. It is less constraining than aclamp or is a strap or a band because it does not need to surround thesite of interest on the body part. This apparatus is more comfortablethan other apparatus currently used to apply force to the skin. Unlike aclamp or a strap or a band, this apparatus will not cause blood vesselsto collapse. Because the apparatus requires access to only one surfaceof a body part of a subject, it can be applied to virtually any site forobtaining samples of interstitial fluids, such as the arm, thigh, orwaist, without any modifications.

Turning now to FIGS. 10A and 10B, the apparatus 100 comprises a cylinder102 and a piston 104. The piston 104 comprises a seal 106 and a pressurehead 108. The pressure head 108 has a bottom portion 109, which has asmall aperture 110 at the lowermost point thereof. The pressure head 108also contains a reservoir 112. The cylinder 102 has a vacuum port 114.The purpose of the cylinder 102 is to position the apparatus over thesite from which interstitial fluids are to be collected. The purpose ofthe piston 104 is to apply sufficient force to the skin to causeinterstitial fluids to emerge therefrom. The purpose of the seal 106 isto maintain the vacuum at a level sufficient for causing the piston 104to apply sufficient pressure to the skin. The purpose of the pressurehead 108 is to provide contact with the skin at the point of applicationof force. In addition, the pressure head 108 has a small aperture 110,through which the interstitial fluid can flow for collection in thereservoir 112.

In operation, a breach is formed in the stratum corneum by one of thetechniques described previously. The apparatus 100 is placed over thebreach, with the cylinder 102 being in contact with the skin so that theaperture 110 is in register with the breach in the stratum corneum. Thevacuum is applied via a pump or the like (not shown) through the vacuumport 114. Under the influence of vacuum, the piston 104 is caused totravel downwards against the skin because of atmospheric pressure actingon the upper surface 116 of the piston 104. See FIG. 10B. The positivepressure exerted on the skin by the pressure head 108 causesinterstitial fluids to flow through the breach in the stratum corneumand through the aperture 110 and collect in the reservoir 112. The fluidcan then be analyzed determine the concentration of analyte.Alternatively, the apparatus 100 is placed over the skin, with thecylinder 102 being in contact with the skin. A breach is then formed inthe stratum corneum so that the aperture 110 is in register with thebreach in the stratum corneum. If desired, pressure can be applied tothe skin prior to forming the breach in the stratum corneum. The vacuumis applied via a pump or the like (not shown) through the vacuum port114. Under the influence of vacuum, the piston 104 is caused to traveldownwards against the skin because of atmospheric pressure acting on theupper surface 116 of the piston 104. See FIG. 10B. The positive pressureexerted on the skin by the pressure head 108 causes interstitial fluidsto flow through the breach in the stratum corneum and through theaperture 110 and collect in the reservoir 112. The fluid can then beanalyzed determine the concentration of analyte. Variations of thesespecific procedures can also be used.

A pressure cuff can be used to apply force and pressure to a body partin which a breach of the stratum corneum has been formed so thatinterstitial fluids can be collected from the breach. In appearance, thepressure cuff is substantially similar to the pressure cuffs used tomeasure a person's blood pressure. In other words, the pressure cuffcomprises a strap or band that is designed to surround the site ofinterest on the body part. Referring now to FIGS. 11A and 11B, apressure cuff 200 comprises a band 202 to which is attached a pressurehead 204. The purpose of the pressure head 204 is to provide contactwith the skin at the point of application of force. At one end of thepressure head 204 are means 206 for attaching the pressure head 204 tothe band 202. Such means 206 may include threads; preferably the threadsare on the exterior wall 208 of the pressure head 204, although thethreads may also be along the interior wall 210 of the pressure head204. The band 202 comprises a means 211 for securing the pressure head204. If the pressure head 204 utilizes threads, the securing means 211preferably also uses threads. The interior of the pressure head 204forms a reservoir 212. At the end opposite the means 206 for attachingthe head 204 to the band 202 is a bottom portion 214 which may becircular, elliptical, square, rectangular or other shape. An aperture216 is formed through the bottom portion 214 to form a communicationchannel to the reservoir 212.

In operation, a breach is formed in the stratum corneum of the bodypart, preferably the forearm, by one of the techniques describedpreviously. The band 202 is placed around the body part so that thepressure head 204 is directly over the breach, so that the aperture 216is in register with the breach in the stratum corneum. The band 202 hasan end 218, which is inserted through a buckle 220. The end 218 of theband 202 can be pulled to tighten the band 202 around the site ofinterest on the body part. The band 202 can be tightened further byincreasing the pressure within a bladder 222, located on the band 202.The pressure can be increased in the bladder 222 by supplying air from apump 224. The increase in pressure can be monitored by a pressure gauge226. The band 202 should be tightened sufficiently so that the pressurehead 204 applies a force to the skin sufficient to cause interstitialfluids to flow through the breach in the stratum corneum and through theaperture 216 and collect in the reservoir 212. The fluid can then beanalyzed determine the concentration of an analyte.

Alternatively, the band 202 is placed around the body part. A breach isformed in the stratum corneum of the body part, preferably the forearm,so that the aperture 216 is in register with the breach in the stratumcorneum. If desired, pressure can be applied to the skin prior toforming the breach in the stratum corneum. The end 218 of the band 202can be pulled to tighten the band 202 around the site of interest on thebody part. The band 202 can be tightened further by increasing thepressure within a bladder 222, located on the band 202. The pressure canbe increased in the bladder 222 by supplying air from a pump 224. Theincrease in pressure can be monitored by a pressure gauge 226. The band202 should be tightened sufficiently so that the pressure head 204applies a force to the skin sufficient to cause interstitial fluids toflow through the breach in the stratum corneum and through the aperture216 and collect in the reservoir 212. The fluid can then be analyzeddetermine the concentration of analyte. Variations of these specificprocedures can also be used.

Upon reading and understanding the invention disclosed herein, it shouldbe apparent to those of skill in the art that modifications and changesto the apparatus and methods disclosed herein can be made while stillfalling within the scope and spirit of the present invention. All suchmodifications and changes are included herein and the invention shouldbe considered limited only by the claims which follow hereafter.

What is claimed is:
 1. An apparatus for the collection of interstitialfluids from the body of an animal, comprising: a cylinder; a pistoncapable of traveling upwardly and downwardly in said cylinder, saidpiston having an upper surface and a pressure head, said pressure headhaving an aperture extending therethrough and in fluid communicationwith a reservoir, said aperture having an area less than the area ofsaid pressure head; a means for evacuating air from said cylinder, sothat said piston can be forced to move downwardly by means ofatmospheric pressure acting on said upper surface of said piston,whereby said piston is capable of exerting a positive pressure on asurface.
 2. A method for the collection of interstitial fluids from thebody of an animal, comprising the steps of: forming a breach through thestratum corneum of the animal, said breach extending at least into theepidermal layer of the skin of the animal; placing the apparatus ofclaim 1 adjacent to said breach in said stratum corneum in such a mannerthat said aperture of said pressure head of said apparatus is inregister with said breach; evacuating air from said cylinder of saidapparatus of claim 1, whereby atmospheric pressure exerts a positivepressure adjacent to said breach in a direction generally toward theskin of the animal; and collecting fluids from said breach.
 3. Themethod of claim 2, wherein the step of forming a breach comprisesforming at least one micropore through said stratum corneum.
 4. Themethod of claim 2, wherein the positive pressure exerted involves aforce ranging from about 1 to about 11 pounds.
 5. A method for thecollection of interstitial fluids from the body of an animal, comprisingthe steps of: placing the apparatus of claim 1 against the skin of saidanimal; forming a breach through the stratum corneum of the animal, saidbreach extending at least into the epidermal layer of the skin of theanimal, said breach being in register with said aperture of saidpressure head of said apparatus; evacuating air from said cylinder ofsaid apparatus of claim 1, whereby atmospheric pressure exerts apositive pressure adjacent to said breach in a direction generallytoward the skin of the animal; and collecting fluids from said breach.6. The method of claim 2, wherein the step of forming a breach comprisesforming at least one micropore through said stratum corneum.
 7. Themethod of claim 2, wherein the positive pressure exerted involves aforce ranging from about 1 to about 11 pounds.
 8. The method of claim 2,wherein said pressure head applies a force prior to the formation ofsaid breach.
 9. An apparatus for the collection of interstitial fluidsfrom the body of an animal, comprising: (a) a band that can be placedaround a body part; (b) a pressure head; (c) a means for securing thepressure head to said band.
 10. The apparatus of claim 9, furthercomprising means for increasing the tightness of said band when saidband is placed around said body part.
 11. The apparatus of claim 10,wherein said means comprises a bladder located on said band.
 12. Amethod for the collection of interstitial fluids from the body of ananimal, comprising the steps of: forming a breach through the stratumcorneum of a body part of the is animal, said breach extending at leastinto the epidermal layer of the skin of the animal; placing theapparatus of claim 9 around the body part of the animal so that saidpressure head is adjacent to said breach; exerting a positive pressureon said pressure head in a direction generally toward the skin of theanimal; and collecting fluids from said breach.
 13. The method of claim12, wherein the step of forming a breach comprises forming at least onemicropore through said stratum corneum.
 14. The method of claim 12,wherein the pressure head applies a force ranging from about 1 to about11 pounds.
 15. A method for the collection of interstitial fluids fromthe body of an animal, comprising the steps of: placing the apparatus ofclaim 9 around the body part of an animal; forming a breach through thestratum corneum of the animal, said breach extending at least into theepidermal layer of the skin of the animal; exerting a positive pressureon said pressure head in a direction generally toward the skin of theanimal; and collecting fluids from said breach.
 16. The method of claim15, wherein the step of forming a breach comprises forming at least onemicropore through said stratum corneum.
 17. The method of claim 15,wherein the positive pressure exerted involves a force ranging fromabout 1 to about 11 pounds.
 18. The method of claim 15, wherein saidpressure head applies a force prior to the formation of said breach.